5-Hydroxytryptamine receptors that facilitate excitatory neuromuscular transmission in the guinea-pig isolated detrusor muscle.

1. In isolated detrusor strips from the guinea-pig urinary bladder, contractile responses to electrical field stimulation were mostly mediated by neurally released acetylcholine (ACh) and adenosine 5'-triphosphate (ATP). 2. 5-Hydroxytryptamine (5-HT) produced a concentration-dependent increase in the amplitude of stimulated detrusor strip contractions. The 5-HT concentration-response curve showed a biphasic profile: the high potency phase was obtained at sub-micromolar concentrations (10-300 nM), while the low potency phase in the range 1-30 microM. The maximum response of the first phase was 30% of the total 5-HT response. 3. Like 5-HT, the 5-HT3 receptor agonist, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT: 0.3-100 microM), the 5-HT2 receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI: 30 nM-3 microM) and the 5-HT4 receptor agonist, 5-methoxytryptamine (5-MeOT: 0.1-30 microM) potentiated, though with lower potency, detrusor contractions. The resulting concentration-response curves were monophasic in nature. 2-Methyl-5-HT had a maximum effect comparable to that of 5-HT. By contrast, the maximal effects of DOI and 5-MeOT were only 20% and 30% of that elicited by 30 microM 5-HT, respectively. 4. The 5-HT3 receptor antagonist, granisetron (0.3 microM) had no effect on the high potency phase, but caused a rightward parallel shift of the low potency phase of the 5-HT curve (pKB = 7.3). Granisetron(0.3 microM) antagonized with comparable affinity (pKB = 7.1) 5-HT-induced responses after pharmacological isolation of 5-HT3 receptors with the 5-HT1/5-HT2 receptor antagonist, methiothepin (0.3 microM) and the 5-HT4 receptor antagonist, GR 125487 (30 nM). Granisetron (0.1, 0.3 and 1 microM) competitively antagonized the potentiating effect of 2-methyl-5-HT with an estimated pA2 of 7.3.5. Methiothepin (0.3 microM) and the 5-HT2A receptor antagonist, ketanserin (0.3 microM) produced a slight inhibition of the first phase of the 5-HT curve. In the presence of ketanserin, an equimolar concentration of methiothepin was ineffective in further reducing the effect of 5-HT. Similarly, the 5-HT4 receptor antagonist, GR 125487 (30 nM) slightly inhibited the first phase of the 5-HT curve. Conversely, this phase was suppressed when detrusor strips were coincubated with ketanserin (or methiothepin) and GR125487.6. In a separate set of experiments, the interactions of 5-HT with either the purinergic or cholinergic components of excitatory neuromuscular transmission were investigated. In the presence of hyoscine(1 microM), 5-HT was mostly effective at sub-micromolar concentrations, while in the presence of the P2-purinoceptor antagonist, suramin (300 microM), 5-HT-induced potentiation was mainly obtained with micromolar concentrations.7. Thus, in electrically stimulated detrusor strips from guinea-pig, 5-HT potentiated excitatory neuromuscular transmission by activating at least three separate neural 5-HT receptors. These include the 5-HT2A and 5-HT4 receptors, which mediate the 5-HT high potency phase mainly by activation of purinergic transmission. On the other hand, the potentiating effect caused by micromolar concentrations of 5-HT mostly involves cholinergic transmission and is mediated by the 5-HT3 receptors.

Characterization of the 5-HT receptor potentiating neuromuscular cholinergic transmission in strips of human isolated detrusor muscle.

In human isolated detrusor strips, submaximal contractile responses evoked by electrical stimulation were resistant to hexamethonium (30 microM) and abolished by tetrodotoxin (0.6 microM) and hyoscine (1 microM), indicating the activation of postganglionic cholinergic nerves. In methysergide (1 microM) and ondansetron (3 microM) pretreated tissues, 5-hydroxytryptamine (5-HT) (0.3 nM-1 microM) caused a concentration-dependent increase in the amplitude of contractions (pEC50 = 8.1), which was antagonized by the selective 5-HT4 receptor antagonist GR 113808 (3, 10 and 30 nM) in a competitive manner. Schild analysis yielded a pA2 estimate of 8.9, a value comparable to that reported for GR 113808 in other animal and human peripheral tissues (8.8-9.7). Our findings indicate that neuromuscular cholinergic transmission in human isolated detrusor muscle is facilitated by neural 5-HT receptors belonging to the 5-HT4 subtype. The human urinary bladder can thus be regarded as an additional site in which 5-HT4 receptors are distributed.

Neural 5-HT4 receptors in the human isolated detrusor muscle: effects of indole, benzimidazolone and substituted benzamide agonists and antagonists.

1. In strips of human isolated detrusor muscle, the 5-hydroxytryptamine (5-HT) receptor (5-HT4) that mediates facilitation of neuromuscular cholinergic transmission was further characterized by using 5-HT and a series of ligands known for their 5-HT4 agonist (5-methoxytryptamine: 5-MeOT, cisapride, (R,S)-zacopride, BIMU 8) or antagonist (RS 23597, GR 125487, DAU 6285) properties. 2. In the presence of methysergide (1 microM) and ondansetron (3 microM) to isolate pharmacologically the 5-HT4 receptors, 5-HT (0.3 nM-1 microM), 5-MeOT (10 nM -30 microM), BIMU 8 (10 nM-3 microM), cisapride (0.1-10 microM) and (R,S)-zacopride (0.1-30 microM) potentiated cholinergic contractions to electrical field stimulation in a concentration-dependent manner. RS 23597 (10 nM-10 microM), a competitive 5-HT4 receptor antagonist in other systems, also showed agonist properties. The following rank order of potency as an agonist was obtained: 5-HT (pEC50 = 8.0) > RS 23597 (7.0) = BIMU 8 (6.9) > or = cisapride (6.6) > 5-MeOT (6.0) > or = (R,S)-zacopride (5.7). Relative to 5-HT (intrinsic activity = 1), 5-MeOT acted as a full agonist (1.03), while BIMU 8 (0.76), (R,S)-zacopride (0.61), RS 23597 (0.60) and cisapride (0.41) behaved as partial agonists. 3. The potentiation by 5-HT was competitively antagonized by the selective 5-HT4 receptor antagonist GR 125487 (0.3-3 nM) with a pA2 estimate of 9.75 (Schild slope of 1.09), and by DAU 6285 (1 microM; pK3 = 6.45). Additionally, GR 125487 (3 nM) antagonized the responses to 5-MeOT (pKB = 9.72) and reversed the potentiation induced by RS 23597. As an antagonist, RS 23597 (10, 30 and 100 nM) inhibited the response to 5-HT. In addition, 30 and 100 nM RS 23597 reduced the 5-HT response maximum by 30 and 50%, respectively. The pKB value calculated at 10 nM was 8.0. 4. Thus, in the human isolated detrusor muscle, the 5-HT4 receptors mediating facilitation of cholinergic neuromuscular transmission are activated by indoleamines (5-HT, 5-MeOT), substituted benzamide (cisapride, (R,S)-zacopride), benzoate (RS 23597) and benzimidazolone (BIMU 8) derivatives. The activities (in terms of both potency and efficacy) of most agonists, as well as the affinity estimates of the antagonists GR 125487 and DAU 6285, are comparable to those found in other peripheral tissues. Exceptions are RS 23597, which acted either as a partial agonist or as an antagonist of the response to 5-HT1 and 5-MeOT that showed an unusually low potency. The latter findings may be ascribed to differences in the efficiency of receptor coupling mechanisms and/or in the molecular structure (i.e. splice variants) of the 5-HT4 receptor.

Signalling mechanism coupled to 5-hydroxytryptamine4 receptor-mediated facilitation of fast synaptic transmission in the guinea-pig ileum myenteric plexus.

5-hydroxytryptamine (HT)4 receptor agonists stimulate gastrointestinal motility partly by facilitating acetylcholine release from myenteric neurones. However, the signalling mechanisms that couple 5-HT4 receptor activation to increased transmitter release in the myenteric plexus are unknown. We used conventional intracellular electrophysiological methods to record fast excitatory postsynaptic potentials (fEPSPs) from neurones in the guinea-pig ileum myenteric plexus preparation. The substituted benzamide, renzapride, acted at 5-HT4 receptors to facilitate fEPSPs. This response was mimicked by forskolin, an activator of adenylate cyclase. Facilitation of fEPSPs by renzapride and forskolin was not blocked by treating tissues with pertussis toxin (PTX) (2 h, 2 microg mL-1). Facilitation of fEPSPs caused by renzapride was blocked by the non-selective protein kinase inhibitors, staurosporine (1 micromol L-1) and H-8 (30 micromol L-1) and by the selective protein kinase A (PKA) inhibitor, H-89 (10 micromol L-1). These data indicate that 5-HT4 receptors act via a PTX-resistant mechanism to activate PKA. Protein kinase A activation leads to an increase in transmitter release from myenteric nerve terminals and a facilitation of fast excitatory synaptic transmission.

5-HT3 receptor involvement in descending reflex relaxation in the rabbit isolated distal colon.

In whole segments of rabbit distal colon with mucosa removed, descending reflex relaxations of the circular muscle (descending inhibition) elicited by inflating (0.1-1 ml) an intraluminal balloon, were partially antagonized by 100 microM hexamethonium and the 5-HT3 receptor antagonist, ondansetron (3 microM), and abolished by 1 microM tetrodotoxin. The inhibitory effects of hexamethonium and ondansetron were additive. Conversely, hexamethonium (100 microM) and ondansetron (3 microM) failed to reduce electrically induced non-adrenergic non-cholinergic (NANC) relaxations of colonic circular muscle. It is concluded that interneuronally released acetylcholine and 5-hydroxytryptamine (5-HT) activate descending inhibitory pathways supplying the circular coat, via nicotinic and 5-HT3 receptors, respectively. This evidence suggests a functional involvement of 5-hydroxytryptaminergic transmission in the descending inhibition of rabbit colon.

Purinergic fast excitatory postsynaptic potentials in myenteric neurons of guinea pig: distribution and pharmacology.

BACKGROUND & AIMS: Adenosine triphosphate (ATP) acting at P2 receptors mediates some fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons of guinea pig ileum. The present studies investigate the distribution of purinergic fEPSPs along the length of the gut and characterize the P2-receptor subtype mediating fEPSPs. METHODS: Conventional intracellular electrophysiological methods were used to record from myenteric neurons in vitro. RESULTS: At a membrane potential of -97 +/- 1 mV, the amplitude (25 +/- 1 mV; n = 307) of fEPSPs was similar along the gut. Hexamethonium (100 micromol/L) inhibited fEPSPs in the gastric corpus by 98% +/- 1% (n = 31) and in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 42%-55%. In the presence of hexamethonium, suramin (100 micromol/L) or the P2X antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 micromol/L) reduced the control fEPSP amplitude in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 71%-84%. The pharmacology of the purinergic fEPSPs was investigated in detail in the ileum. Noncholinergic fEPSPs were concentration-dependently (1-30 micromol/L) inhibited by PPADS (50%-inhibitory concentration, 3 micromol/L). In addition, alpha,beta-methylene 5'-adenosine triphosphate (1 micromol/L) also reduced purinergic fEPSPs. CONCLUSIONS: Fast EPSPs mediated in part through P2X receptors are prominent in myenteric neurons along the small and large intestines but are rare in the gastric corpus.

Role of nitric oxide-dependent and -independent mechanisms in peristalsis and accommodation in the rabbit distal colon.

Previous findings, which have been confirmed in this study, indicate that nitric oxide (NO)-dependent and -independent (apamin-sensitive) mechanisms underlie the electrically induced non-adrenergic, noncholinergic (NANC) relaxation in the circular muscle of rabbit distal colon. Based on this evidence, we further investigated whether, and to what extent, these separate NANC components participate in the maintenance of circular muscle tone, reflex relaxations evoked by localized balloon distension or during peristalsis (descending inhibition) and in the accommodation of colonic wall in response to graded intraluminal fluid delivery. NG-nitro-L-arginine (L-NNA) (10-300 microM), apamin (100 nM) and tetrodotoxin (60 nM), enhanced the spontaneous low tone and phasic activity in circular muscle strips. In experiments on peristalsis, L-NNA (30 and 300 microM) shortened the latency of peristaltic wave initiation and increased the velocity of propulsion of an intraluminally distended balloon (range, 0.1-1 ml). The latter effect was mimicked by the NO scavenger oxyhemoglobin (30 microM). Velocity of propulsion was enhanced by apamin (100 nM) at low balloon distension (0.1 and 0.2 ml), whereas it was reduced at high distension volumes (1 ml), due to disruption of descending inhibition. A combination of L-NNA (300 microM) and apamin (100 nM) blocked peristalsis, due to persisting spasms of the circular muscle. L-NNA (300 microM) did not affect the amplitude of distension-evoked ascending reflex contraction and slightly inhibited the descending reflex relaxation. By contrast, the latter reflex was virtually abolished by apamin (100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Visual evoked potentials in encephalopathy induced by galactosamine, ammonia, dimethyldisulfide, and octanoic acid.

Visual evoked potentials were utilized to examine the neuronal transmission changes provoked by galactosamine-induced hepatic encephalopathy and by administration in normal animals of toxins presumably involved in the pathogenesis of hepatic encepalopathy. Separate acute administrations of ammonia, dimethyldisulfide, and octanoic acid induced lethargy, convulsions in the case of the first two, and coma with visual-evoked potential patterns that never resembled the evoked potentials recorded in hepatic coma. By contrast, single and repeated administrations of the three above-mentioned toxins together at lower doses induced lethargy and coma with visual-evoked potential patterns similar to those observed in galactosamine-induced hepatic coma. These observations, together with previously published data, are consistent with the concept that the synergistic interaction of these toxins plays a significant role in the pathogenesis of hepatic encephalopathy.

Therapeutic potential of drugs with mixed 5-HT4 agonist/5-HT3 antagonist action in the control of emesis.

Drugs interacting with serotonin (5-hydroxytryptamine, 5-HT) receptors are of value in the treatment of several gastrointestinal disturbances. Selective 5-HT3 receptor antagonists (ondansetron, granisetron, tropisetron) are widely utilized to control emesis induced by chemotherapy and radiation, while agonists at 5-HT4 receptors (cisapride, renzapride, BIMU compounds) are endowed with gastrointestinal prokinetic action. Here we overview the therapeutic potential of drugs with potent mixed 5-HT4 agonist/5-HT3 antagonist properties (i.e. BIMU 1) in the management of anticancer therapy-induced emesis and of intestinal adynamic post-operative conditions associated with vomiting. In the former situation, the agonism at 5-HT4 receptors is expected to be of benefit via two possible mechanism: (i) inhibition of 5-HT release from enterochromaffin cells; (ii) restoration of anally driven peristaltic waves in the upper gastrointestinal tract. Moreover, 5-HT4 receptor-induced prokinetic activity may counteract colonic constipation, an unwanted effect which occurs in a number of patients treated with pure 5-HT3 receptor antagonists. Additionally, the above mentioned drugs might be of value in post-operative conditions associated with intestinal adynamia and emesis sensitive to 5-HT3 receptor blockade.

Effects of mercuric chloride and methyl mercury on cholinergic neuromuscular transmission in the guinea-pig ileum.

The effects of mercuric chloride (HgCl2) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.33 microM slightly enhanced the amplitude of twitch contractions in approximately 50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 microM mercurials increased electrically-evoked tritium outflow from [3H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl2 IC50 = 21.3 +/- 6.4 microM; MeHg IC50 = 45.1 +/- 5.5 microM), as well as contractions to exogenous ACh (0.1 microM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl2 on basal tone was antagonized by the Ca2+ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca2+ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca(2+)-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.

Visual evoked potential: a diagnostic tool for the assessment of hepatic encephalopathy.

Visual evoked potential recordings were examined in 45 liver cirrhosis patients with (n = 29) and without (n = 16) encephalopathy, in 15 normal volunteers, and in one patient with an opioid induced stupor state. Visual evoked potential parameters were classified on the basis of EEG recordings. Plasma concentrations of amino acids, octopamine, and ammonia were assayed in order to document the metabolic change of hepatic encephalopathy. Latencies and wave patterns recorded after flash stimulation differentiated the four degrees of the coma one from another according to EEG classification in the 29 patients with encephalopathy. In the group of 16 patients without clinical and EEG evidence of encephalopathy the visual potential recordings discriminated a group of patients (n = 10) in a preclinical stage of encephalopathy. Biochemical parameters and subsequent clinical observation of patients confirmed our judgement of a preclinical stage of encephalopathy. These results suggest that visual evoked potentials are a simple, suitable and objective method for differentiating the degrees of encephalopathy and for identifying the preclinical stage of encephalopathy.